Applications of plant Genetic Engineering

KOMAL JAMIM SHEHZADI

Asim FarooqBasma Sarfraz

ASAB; NUST

Goals of Genetic Enginnering

Improvement of agronomically important crops.

Production of oils and lubricants.Modification of lipid composition of seed

crops. Mnufacturing of industrially important

chemicals.And pharmeceutically active compounds. Development of stress tolerant plants.

Genetically Engineered traits (Big Six)

Six genetically traits which areHerbicide resistanceInsect resistanceViruses resistanceAltered oil contentsDelayed fruit ripeningpollen control

Herbicides resistance

• Weeds reduces the yields.• Compete for nutrients,sunlight,and water

etc.• Herbicides reduces the impact of weeds.

Mode of action of herbicides Herbicides disrupts disrupt one or

more metabolic pathway or other vital processes to kill the weeds.

Disadvantage of using Herbicides

• Weeds still reduce crop productivity by approximately 12%.

• Some traditional crops are more sensitive to herbicides.

Solution

• Modification of crops with foreign genes.• Reduction of number of

application of herbicides.• Using only single chemical.• Using biodegradable or less

toxic herbicides.

Critics

• If chemical approach to weed control continues there will be

Incresed amount of chemicals in our environment.

Incresed dependency on toxic chemicals

Weeds may become resistance to herbicides.

Types of GE herbicides resistance• There are two types

1)modification of target (protein) so that it no longer binds to herbicides.

2) production of new protein that inactivates the herbicides.

Herbicides and resistance modified

crops• Crops plants have been engineered to be resistance to four herbicides.

Herbicide: Resistance modified crops

Glyphosate: soyabeans,corn,canola,cotton

and sugarbeets Glufosinate:

soyabeans,canola,cotton,corn sugarbeets rice. Bromoxynil: cotton sulfonlurea: cotton and flax

Glyphosate resistance

• Most commonly used herbicide.Known as Roundup

Mode of Action Inhibits EPSPS (5-enolpyruvylshikimate-3-

phosphate synthase) in the shikimate pathways in both plants and bacteria.

Gene encoding EPSPS in a glyphosate resistance E.coli strain was isolated.

Under control of plant promoter transferred into plant cells.

e.g soyabeans,corn,canola and cotton.

Glufosinate resistance

• Market name Basta,and Liberty. Mode of ActionActive ingredient (phosphinothricin)

in glufosinate mimics the structure of glutamine.

This blocks the activity of glutamine synthase(required for nitrogen metabolism).

Gene is isolated from the bacterium streptomyces,whose protein products inactivates phosphinothricine in the herbicide.

e.g soyabean, corn.

Bromoxynil resistance

• Nitrile herbicide marketed as Buctril,and bronate.

Mode of ActionInactivates photosynthesis in

plants.Bromoxnil resistance plants are

producedby transferring the gene encoding the enzyme bromoxnil nitrilase (BXN).

Gene is obtained from soil bacterium klebsiella pneumoniae.

Nitrilase inactivates bromoxynil(e.g cotton).

Mode of Action

Sulfonlurea

• Environmentally friendly herbicide.Discovered by Dupont crop protection in 1975.

Mode of ActionBlocks an enzyme acetolactase

required for the synthesis of amino acid(valine,lucine,and isolucine).

Herbicide resistance is developed by modifying the enzyme by gene mutation and then transferred into plants.

e.g cotton.

Non Resistant Resistant

Insect Resistance Biopesticides Bt-based insect resistance; successfully engineered Bt gene into different plant varieties Protease inhibitors PI’s are present in Storage Tissues contributing to the total of 10% of protein content (Huma and Khalid, DOB, UOK, Aug, 2007) First observed in larvae growing on soya bean products Transgenic plants produced; cowpea trypsin inhibitor gene, potato protease inhibitor, α-amylase inhibitor One gene killing more insects in different plants

Continued…. Resistance of insects against Bt toxin

and PIs Two methods of PI resistanceHigh level expression of soybean-

trypsin-inhibitor gene in transgenic tobacco plants failed to confer resistance against Helicoverpa armigera

Delaying insect resistance to transgenic crops

High dosage refuge strategy can be used to somehow delay the process of insect resistance (Bruce E.T. Department of Entomology, University Of Arizona)

Virus Resistance Every year, tons of plants and plant

products are lost due to viral infections Different chemicals supplied to kill

viral vectors Viral DNA multiplies within the cell,

thousands of copies are produced, virus infects next cell(s)/Plant(s)

Two genetic techniques are involved: Coat protein-mediated resistance RNA mediated virus resistance

Coat Protein-Mediated Resistance Plant is genetically engineered with a

particular viral coat protein gene Roger N. Beachy from Donald Danforth

Plant Science centre, St. Louis, USA carried out studies on TMV an reported that transgenically expressed CP interferes with disassembly of TMV particles in the inoculated transgenic cell. Using the known coordinates of the three–dimensional structure of TMV, mutant forms of CP were developed that had stronger inter–subunit interactions, and confer increased levels of CP–MR compared with wild–type CP

Continued… Numerous transgenic plant varieties

have been produced for resistance against Cucumber mosaic virus, alfalfa mosaic virus, tobacco rattle virus etc.

Resistance genes for a many other disease causing agents like fungi and bacteria have also been produced

Genes from tomato have been isolated against P. syringae and C. fulvum. A flax gene develops resistance against fungal rust disease

Transgenic varieties of Yellow Squash and Papaya

RNA-Mediated Virus Resistance Directs the used of genetically altered

RNA to stop viral multiplication within the cell

49 plants were generated that express the 5' untranslated region of the tobacco etch potyvirus (TEV) genome ligated to a mutated version of the TEV coat protein gene sequence that rendered it untranslatable. Eight different transgenic plant lines were analyzed for transgene expression and for resistance to TEV (Dougherty et al, DOM, Oregon State University)

Altered Oil Content Various types of oils are produced in

plants which differ due to difference in the fatty acid content etc

Plant oils are largely used commercially as well as domestically

Genes with altered properties of improved oil traits are cloned into soybean. These genes are selected through the particular gene promoters.

Reduced palmitic acid content, 80% oleic acid and 30% stearic acid content

Considerable change in fatty acid content

Delayed Fruit Ripening Basically a hormone regulated process Genetically engineered tomatoes were

marketed, the only delayed fruit of the times

Technique not much developed The ACC synthase gene obtained from

fruits was engineered in antisense form in the papaya tissue through the particle gun. The gene translated to protein which resulted in delayed fruit ripening (Evelyn Mae, IPB, University of Philippines)

Flavr Savr Tomato

First genetically modified food Polygalacturonase gene was blocked Resulted in delayed ripening of tomato Antisense gene technology was used FDA approved in May 1994 Produced only for 1 year

Pollen Control Due to improved qualities, hybrid crops are used

in agriculture Hybridization must be in a controlled manner Male flower part is removed before pollen

release Production of male sterile plants by inserting a

gene from Bacillus amyloliquefaciens , producing barnase

Barstar gene technique is also used Barstar pollen fertilizes male sterile barnase

plant and a fertile hybrid is produced Corn and chicory have been genetically

engineered through this technique

• The initial objective was to improve crop protection.

1. through the introduction of resistance against plant diseases.

2. through increased tolerance towards herbicides.

GE foods- a big splash…

Controversies generated…

• Few incidents:• In 1989, GE supplement L-tryptophan… outbreak of

Eosinophilia Myalgia Syndrome (EMS)… 5000 people suffered.

• In 1999, GA potatoes… poisonous to mammals… when fed to rats… caused highly weight reduction of many organs, impairment of immunological responsiveness and signs suggestive of viral infection.

• Cornell University scientists reported recently that 44% of monarch butterfly larvae died within

four days when fed milkweed that had been dusted with pollen from GA corn.

Potential risks to human health

• The safety assessment of GM foods generally investigates:(a) direct health effects (toxicity).(b) tendencies to provoke allergic reaction (allergenicity).(c) specific components thought to have nutritional or toxic

properties.(d) the stability of the inserted gene.(e) nutritional effects associated with genetic modification.(f) any unintended effects which could result from the gene insertion.

SOYBEAN…• The first genetically modified soybeans were planted in the

US in 1996, that was Roundup Ready (RR) soybean, was developed by the biotech giant Monsanto.

• In 2007, 216 million tones of soybeans were produced worldwide. The world’s leading soybean producers are the US(33%), Brazil (27%), Argentina (21%), and China (7%).

• Each year, the EU imports approximately 40 million tones of raw soy products, primarily destined for use as cattle, swine, and chicken feed.

• In addition, soybeans are used to produce numerous food ingredients , oils and additives. Lecithin, for example, is used as an emulsifier in

chocolate, ice cream, margarine, and baked goods.

Yield enhanced…• Even a small change in the amount of oil a soybean

produces could affect industry profits marginally, the main aim was creating higher oil content without disrupting protein content.

• Enhancement of soybean oil content was achieved by the introduction of a seed-specific transgenic for a DGAT2-type enzyme from the oil-accumulating fungus Umbelopsis ramanniana.

• In these studies, the oil content was increased from approximately 20% of the seed weight to approximately 21.5%.

DuPont and Monsanto• The high-oleic soybean, developed by DuPont

is one of the first in a wave of bioengineered cash crops that are being altered for nutritional purposes by removing primary antigen( protein P34) from soybean.

• Currently, Monsanto has also introduced two varieties of biotech soybeans.

1. By promising to eliminate trans fats.2.And produce oil with omega-3 fatty acid for

use in yogurt, granola bars and spreads.

Disadvantage

• For the first time since 1996, acres of Roundup Ready genetically modified soybeans could drop as more farmers decide to plant non-GMO.

• “Farmers upset with Monsanto”.• Hamsters Experiment showed devastating

results- made upset.

Conclusion

• GM foods have the potential to solve many of the world's hunger and malnutrition problems.

• It helps protect and preserve the environment by increasing yield and reducing reliance upon chemical pesticides and herbicides.

• Yet there are many challenges ahead for governments, especially in the areas of safety testing, regulation, international policy and food labeling.

CORN• Genetically engineered corn was first sold in 1996,

and since then, scientists have introduced a variety of new genes.

• In 2002, 32% of field in US was genetically engineered planted within 12.4 million hectares.

• Most common is Bt insect resistant corn, e.g. from European corn borer and corn root worm.

• Other include resistance to different herbicides. Disease-resistant corn crops may have lower levels of mycotoxins, potentially

carcinogenic compounds.

Disadvantages

• The study found that three strains of molded crops -- MON 810 and MON 863, which are resistant to pests, and NK 603, which is fortified to withstand weed killer -- significantly disrupted the blood chemistry of rats who ate them.

CANOLA

• A genetically engineered rapeseed that is tolerant to herbicide was first introduced to Canada in 1995.

• In 2008 genetically modified (GM) herbicide tolerant canola is being grown commercially in Australia for the first time.

• Today 80% of the acres sown are genetically modified canola.

• In 2003, Australia's gene technology regulator approved the release of canola altered to make it resistant to glufosinate ammonium, a herbicide.

NUTRITIONALLY ENHANCED PLANT“GOLDEN RICE”

• Rice that has been…1. Genetically modified.2. Contain beta carotene.3. Produce vitamin a once consumed.

• Produced by combining genetic material from• Daffodils• Peas• Japonica rice.

Developed by…

• Developed by two scientists in 1999.• Dr. Ingo Potrykus of the Institute for Plant

Sciences at the Swiss Federal Institute of technology in Zurich, Switzerland.

• Dr. Peter Beyer of the center for Applied Bioscience at the University of Freiburg in Germany.

How Does It Work?

• The addition of 2 genes in the rice genome will complete the biosynthetic pathway:

1. Phytoene synthase (psy) – derived from daffodils.2. Lycopene cyclase (crt1) – from soil bacteria Erwinia

uredovora.• Produces enzymes and catalysts for the

biosynthesis of carotenoids (β-carotene) in the endosperm.

• Presence of pro-vitamin A gives rice grains a yellowish-orange color, thus,

the name ‘Golden Rice’.

• V ita m in A : • Sources from: Fish oil, Liver Milk, Eggs Butter,

Orange/Yellow fruits and vegetables. • Required for: Growth Healthy skin & cells, Good night

vision.• Deficiency (VAD): Blindness ,weakened immune system,

increased susceptibility to infection and cancer, anemia, deterioration of the eye tissue, cardiovascular disease and can increase the risk of maternal mortality.

• Facts: Between 100 and 140 million children are vitamin A deficient each year. About 250,000 to 500,000 become blind, (1/2 dying within 12 months of losing sight), 600,000 women die from childbirth-related causes each year.

Limitations

• Vitamin A in “Golden” rice is not sufficient for the daily recommended amount.

• Even if it was enough, people require zinc, proteins, fats and other elements (which they also often lack) in their diets to convert the beta-carotene to vitamin A.

• White rice is associated with culture and tradition ; golden rice may not be accepted .

BENEFITS

• May solve VAD in third world countries. • Can improve amount of Vitamin A. • May influence the acceptance of GMOs if

successful.• Golden Rice may not be the complete solution

to VAD, but it is a start and a contribution to the problem.

• GMOs have actually reduced the use of herbicides and pesticides.

Free Distribution

• Funded by the Rockefeller Foundation, the Swiss Federal Institute of Technology, and Syngenta, a crop protection company.

• Golden Rice Humanitarian Board-responsible for the global development, introduction and free distribution of Golden Rice to target countries.

MOLECULAR FARMING(BIOPHARMING)

• It is the use of genetically engineered crops to produce compounds with therapeutic value , including antibodies, blood products, cytokines, growth factors, hormones, recombinant enzymes and human and veterinary vaccines.

Uses • Several PDP products for the treatment of

human diseases are approaching commercialization, including

1. Recombinant gastric lipase for the treatment of cystic fibrosis.

2. Antibodies for the prevention of dental caries etc.

• Potential products include the development of antigens for vaccines that might be mass-produced in plants such as corn and used to fight such diseases as cancer and diabetes.

Antibodies to HIV• Today in London, UK regulators announced the

approval of Europe’s first clinical trial of an anti-HIV product produced in genetically modified tobacco plants.

• Plant-produced antibody designed to stop transmission of HIV when applied directly to the vaginal cavity.

• The active ingredient is an antibody called P2G12 – it recognizes proteins on the surface of HIV to block infection. More specifically, it’s a monoclonal antibody made from immune cells for one specific role.

Advantages

1. Commercial scale-up involves simply planting seed rather than using costlier fermenters.

2. Plants do not carry pathogens that might be dangerous to human health. 3. On the level of pharmacologically active proteins, there are no proteins

in plants that are similar to human proteins.4. Like animals, plants are complex, multicellular organisms and therefore

their process of protein synthesis is more similar to that of animals than those of bacteria or yeast.

5. Purification of the desired product from plants is often easier than from bacteria, which can be labour and cost intensive.

6. Transgenic plants can be grown on an agricultural scale requiring only water, minerals and sunlight.

7. Plants offer advantages over live animals and animal cell cultures in terms of safety, cost, time involved, and storage and distribution issues.

Expressions SystemExpressions System

Yeast

Bacteria

Plant viruses

Transgenic Plants

Animal Cell Cultures

Transgenic Animals

Cost of maintaining

inexpensive

inexpensive

inexpensive

inexpensive

expensive

expensive

Protein yield

High

Medium

Very high

High

Medium to high

High

Therapeutic risk

Unknown

yes

Unknown

Unknown

yes

yes

Type of storage

-2.0°C

-2.0°C

-2.0°C

RT*

N2**

N/A

Gene size (protein) RestrictionUnknown

Unknown

Limited

Not limited

Limited

Limited

Production cost

Medium

Medium

Low

Low

High

High

Raising industry

• Today Molecular Farming is considered "big business". According to the Canadian Food Inspection Agency, in a recent report, says that U.S. demand alone for biotech pharmaceuticals is expanding at 13 percent annually and to reach a market value of $28.6 billion in 2004. Molecular Farming is expected to be worth $100 billion globally by 2020.

EDIBLE VACCINES

• Defination: A vaccine that is based on the genetically engineered

expression of an antigenic protein by an edible plant. Following consumption, the protein is recognized by the immune system.

• History:• The first journal publication describing edible vaccine was

published in 1992.• The studies was conducted by Mason, Lam and Arntzen at Texas

A&M University.• A tobacco plant was successfully transformed and Hepatitis B

surface antigens was expressed.

Unique Properties• It hold great promise as a cost-effective, easy-to-administer,

easy-to-store, fail-safe and socioculturally readily acceptable vaccine delivery system, especially for the poor developing countries.

• It involves introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins.

• Initially thought to be useful only for preventing infectious diseases, it has also found application in prevention of autoimmune diseases, birth control, cancer therapy etc.

• Edible plant-derived vaccine may lead to a future of safer and more effective immunization.

• Resistance to genetically modified foods may affect the future of edible vaccines.

Currently searched plants vaccine

• Tobacco- Human Papillomavirus.• Peanuts- Rinderpest Virus.• Papaya- Cysticercosis.• Potato- Rotavirus.• Rice- Helicobacter pylori.• Bananas- E .coli, HBV.• Tomato- Malaria virus.

Advantages of edible vaccines

• Humanitarian- save millions of lives.• Cut down costs - vaccines are given orally, no purification of

protein is needed. - elimination of medical professionals.• Renewable resource.• Palatability.

Unsafe but useful…

• The concept is intriguing, in practice it could prove quite unsafe and risky.

• Even the creator of the edible vaccine, Charles Arntzen, now concedes that the idea should be abandoned.

• Research in this area now focuses on non-food crops where the vaccine is subsequently purified and packaged as a pill or capsule. It permits wider distribution and easier storage of the product.

• As a result, developing countries would be capable of distributing much-needed vaccines that are impractical in a liquid form, which requires refrigeration or must be administered via injection.

BIOPOLYMER

• BIOPOLYMERS look exactly like a conventional plastic and have similar characteristics. The difference is just that they are made from vegetable raw materials.

• Biopolymers are produced from annual crops and plants like wheat, corn, potato and sugar cane and other new alternatives now being developed for the production of food packaging materials like film sheet and foam containers. i.e. PLA (Polylactic Acid).

Types of Biopolymer

• Some examples: The input materials for the production of these

polymers may be either renewable (based on agricultural plant or animal products) or synthetic.

• There are four main types of green polymer based respectively on:

• 1. Starch• 2. Sugar• 3.Cellulose• 4.Synthetic materials

Research demonstrated

• Plants are able to produce PHBV using 3 genes from bacterium Alcaligenes eutrophus into canola for synthesis of PHB in seeds.

• Poly b-hydroxybutyrate is produced in experimental plant Arabidopsis.

Applications of green polymers

• Every biopolymer has its own material-specific properties, e.g. barrier properties such as oxygen permeability.

• The barrier properties are relevant to the choice of biopolymers for the packaging of particular products.

• Bioplastics have very promising prospects for use in pesticide soil pins, for packaging in-flight catering products and for packaging dairy products.

• The Biota Water bottle looks and feels like plastic, but it's actually made from corn.

• The new, high-tech material is just as effective as plastic and it's biodegradable.

• If you throw it in a landfill or compost pile, the bottle dissolves in as little as 80 days, according to Biota.